Micro labs - review

BIOL260Winter 2012

Ubiquity

• What organisms grow best at room temperature? ___°C?

• At body temperature? = ___°C?• What kind of medium is TSA?

Appearance of growth on plates

• Bacteria vs fungi (molds)?

Aseptic technique

• What does it mean?• Why do we need to use it?• What does sterile broth look like?

Inoculation of media

• Streaking for isolation – how and why?• Can you tell if growth on a slant is pure

culture? • How do you inoculate a broth?• A slant?• A deep?

Microscope

• What type of microscope did we use for our labs?

Simple and differential stains

• What is the difference?• How do you prepare a slide for staining?

The Gram stain!

• Know the steps including names of all reagents & what their function is

• Know what the staining properties of bacteria tell you about the organisms

• Know what can go wrong

Misc other stains

• Endospores – How do they appear in Gram stain– What is one species of bacteria that forms endospores

• Flagella– How do they appear in the Gram stain?– What result would you expect for a motility test?

• Acid fast bacteria– How do they appear in Gram stain?– What is the primary genus of acid fast bacteria?

Endospores in Gram stain

Endospore stain:Malachite green + safranin

Flagella stain

Acid fast bacteria

Chemically defined media

• Glucose mineral agar = glucose salts agar (GSA)

Complex/undefined media

• Trypticase soy agar (TSA)• Tripticase soy yeast agar (TSY)• Mueller Hinton agar

Selective media

• Antibiotics, toxic dyes, bile and other selective inhibitors of bacterial growth

Differential media

• pH indicators:– Brom cresol

purple– Phenol red

Selective & differential media• Eosin methylene blue (EMB)

– SELECTS for Gram negative rods: eosin is toxic to Gram positive bacteria– DIFFERENTIATES lactose fermentors from non-lactose fermenters: acid

produced during lactose fermentation causes the colonies to appear dark pink or purple

• Mannitol salt agar– SELECTS for salt-tolerant Gram positive cocci = Staphylococcus species– DIFFERENTIATES mannitol fermenters from non-fermenters

• MacConkey agar– SELECTS for Gram-negative rods– DIFFERENTIATES lactose fermenters fron non-fermenters

• LES Endo agar– SELECTS for Gram negative rods– DIFFERENTIATES coliform bacteria (Enterobacteriaceae) from Salmonella

MacConkey agar: lactose fermenters turn the agar yellow

Mannitol salt agar: mannitol fermenters turn the agar yellow

Viable cell count

• Determine the cells/ml in the original stock solution based on a bacterial count plate, given the following:A. 1 ml of a 1:100 dilution (10 -2 dilution) of the

stock culture was added to the count plate and the count plate contains 72 bacterial colonies

B. 0.1 ml of a 1:1000 dilution (10 -3 dilution) of the stock culture was added to the count plate and the count plate contains 115 bacterial colonies

Count plate ex. 1

Number of colonies divided by dilution factor times volume, in ml, of diluted medium that was plated:

= 72 x 1/10-2 x 1ml= 72 x 102 = 7.2 x 103 organisms per ml in stock

culture

Count plate ex. 2

Number of colonies divided by dilution factor times volume, in ml, of diluted medium that was plated:

= 115 x 1/10-3 /0.1ml= 115 x 103 / 0.1ml = 115 x 104 = 1.15 x 106 organisms/ml in stock

culture

Aerobic/anaerobic lab: oxygen requirements

• Aerobes: require oxygen• Obligate anaerobies: require that there NOT

be oxygen• Facultative anaerobes: can grow in either

aerobic or anaerobic conditions• The shake agar/deep tube: what did we use

this for? Do you remember how to read it?

UV light

• What is the effect of UV light on bacteria? • What factors will influence how much damage

is done by UV light?• Why did we incubate the plates in the dark?

UV light

Antimicrobial drugs

• What is the Kirby-Bauer test? • What does it tell you about the organism?• What is a “zone of inhibition”?• What do you need to know in order to

interpret the results of a KB test? What about a disinfectant?

KB test

Transformation lab

• What is transformation?• What was the positive control for this lab?• What was the negative control? Why did we

use controls?• What was the function of the streptomycin in

the TSY plate used in step 2 of this exercise?

Normal skin microbiota

• What types of organisms normally inhabit our skin?

• Which of the normal skin inhabitants can grow in both anaerobic and aerobic conditions? What are these types of organisms called?

• TSY + glucose + brom cresol – what does this medium allow us to determine?

Skin microbiota

• Staphylococcus epidermidis: Gram positive, facultative, coag negative

• Staphylococcus aureus: Gram positive, facultative; coag positive

• Micrococcus luteus: Gram positive coccus, aerobe• Propionibacterium acnes, P. granulosum: Gram

positive, anaerobic coryneform (diptheroid) rod• Bacillus subtilis, B. cereus: Gram positive rods,

facultative

Coagulase test

• Used to differentiate coagulase-producing species of staphyloccus (coagulase-postive staph) from non-coagulase producing species (coagulase-negative staph) – important in differentiating pathogenic from non-pathogenic isolates

Throat culture lab

• What types of organisms are normal inhabitants of your throat?

• What type of plates do you use to observe hemolysis?

• What does the type of hemolysis tell us about an organism?

• What organism causes strep throat? Is it hemolytic? If yes, what type of hemolysis?

Sore throat: is it viral or bacterial?

• If the only bacteria isolated are non-pathogenic, this SUPPORTS it being a viral infection

• It doesn’t CONFIRM it being a viral infection because you have not actually isolated a virus

• Viruses DO NOT grow on bacterial culture plates

Hemolysis

Identification of gram negative rods

• What is phenol red used for? How do you interpret a test that uses this dye as an indicator?

• What is a durham tube?

Differential media: fermentation broths

1: No acid, no gas2A. Weak acid, no gas2B. Strong acid, no gas3A & 3B: Strong acid + gas

Negative urea Positive urea

Water lab

• What are the “indicator organisms”?• MPN test for lactose fermentation• MPN index = ?• + BGLB tubes / LES endo plates – why?

What sugar fermentation profile indicates a fecal coliform?